The present invention relates to improved syntheses of known and novel benzothiazine dioxide which are potent and selective endothelin antagonists. The processes of the instant invention are improved over those recited in U.S. Pat. No. 5,599,811 which is hereby incorporated by reference.
The compounds of the instant invention exhibit very significant improvements over those described in U.S. Pat. No. 5,599,811. These improvements include: binding affinity to the ETA receptor, ETA selectivity, functional activity, long pharmacokinetic half-life, high bioavailability, in vivo activity in inhibiting the pressor effect caused by bET-1, oral activity with relatively long duration of action, and efficacy in acute hypoxic pulmonary hypertension in rats.
The processes of the instant invention provide more facile syntheses with higher yields. They are short, clean, reproducible and no tedious chromatography is needed. Moreover, the processes are scaleable and therefore useful for large-scale development.
The present invention also relates to antagonists of endothelin useful as pharmaceutical agents, to pharmaceutical compositions which include these compounds and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment. More particularly, the compounds of the present invention are antagonists of endothelin useful in treating elevated levels of endothelin, acute and chronic renal failure, essential renovascular malignant and pulmonary hypertension, cerebral infarction and cerebral ischemia, cerebral vasospasm, cirrhosis, septic shock, congestive heart failure, endotoxic shock, subarachnoid hemorrhage, arrhythmias, asthma, preeclampsia, atherosclerotic disorders including Raynaud""s disease and restenosis, angina, cancer, benign prostatic hyperplasia, ischemic disease, gastric mucosal damage, hemorrhagic shock, ischemic bowel disease, and diabetes.
Also, the compounds will be useful in cerebral ischemia or cerebral infarction resulting from a range of conditions such as thromboembolic or hemorrhagic stroke, cerebral vasospasm, head injury, hypoglycemia, cardiac arrest, status epilepticus, perinatal asphyxia, anoxia such as from drowning, pulmonary surgery, and cerebral trauma.
This invention is improved processes for the preparation of compounds of Formula 1 
or a pharmaceutically acceptable salt thereof wherein
R1 is hydrogen, alkyl, or alkoxy;
R2 is hydrogen or alkoxy;
R3 is alkyl or alkoxy;
R2and R3may be joined to form a ring 
R4 is hydrogen or alkyl;
R5 is hydrogen, alkyl, alkoxy, halogen at the 2 or 3, or 4, or 5 positions or R5 is a 3,4-methylenedioxo; and
R6 is CF3, halogen, alkyl, benzyl, phenyl, hydroxy, or pyrrole comprising:
a) alkylating a compound of formula A 
xe2x80x83using sodium hydride in DMF followed by reaction with methyl bromoacetate to produce a compound of formula B 
b) combining compound B in THF with a solution of TiCl4 in solvent at xe2x88x9278xc2x0 C., treating with triethylamine and quenching with an acid to produce a compound of formula C 
c) treating compound C with triflic anhydride in a solvent in the presence of pyridine for from 1 to 5 hours to produce a compound of formula D 
d) coupling the compound D with a boronic acid of formula X 
xe2x80x83in DMF and toluene in the presence of a palladium catalyst and potassium carbonate at about 100xc2x0 C. to produce a compound of Formula 1.
The free acid of Formula 1 is obtained by sponification of the ester with, for example, LiOH in THF/MeOH or in dioxane. Any strongly alkaline solution in methanol can be used.
Compounds of the invention are those prepared by the above process, especially those selected from:
4-(3,5-Dimethoxy-phenyl)-2-(2-trifluoromethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Chloro-phenyl)-4-(3,5-dimethoxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Bromo-phenyl)-4-(3,5-dimethoxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Chloro-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Benzyl-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2,6-Dimethyl-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid monosodium salt;
4-(7-Methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-ethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1 ,3]dioxol-5-yl-2-(2-propyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-isopropyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-butyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-fluoro-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-hydroxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2,3-dichloro-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2,4-dichloro-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-chloro-4-methoxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(3-chloro-2-methyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2,6-dimethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-1,1-dioxo-2-(2-pyrrol-1-yl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(3,4-dimethyl-isoxazol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-(6-Methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid; and
4-(3,5-Dimethyl-phenyl)-2-(2-trifluoromethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid.
The invention is also a process for the preparation of a compound of Formula 1 
comprising:
a) treating aryl bromide with n-butyl lithium followed by zinc bromide to generate an aryl zinc bromide of Formula Y 
b) reacting the product of step a) above with 
xe2x80x83in THF in the presence of a palladium catalyst to produce a compound of Formula 1 
The free acid of Formula 1 is obtained by sponification of the ester.
The following three compounds are obtained by the above process:
2-(2-Bromo-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid monosodium salt;
4-(7-Ethyl-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid, potassium salt; and
4-Benzo[1,3]dioxol-5-yl-2-(2-benzyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid.
The invention is also a process for the preparation of a compound of formula B 
comprising:
a) reacting a phenyl sulfonyl chloride with 1 equivalent of aniline to produce a sulfonamide of formula F 
b) lithiating the product of step a) above at low temperatures and quenching with CO2 to form a compound of formula G 
c) treating the product of step b) above with acetic anhydride and a catalytic amount of methanesulfonic acid to produce a compound of formula H 
d) treating the product of step c) above with NaOMe followed by methyl bromoacetate to produce a compound B as above.
Compound B is carried over to Formula 1 as described before.
Compounds of the invention are those prepared from the above process, especially 4-(7-Ethyl-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester.
The sodium and potassium salts of the above compound are preferred.
The invention is also a process for the preparation of a compound of Formula 1 which comprises
a) refluxing 
xe2x80x83with one equivalent of pyridine and DMAP to produce cyclic intermediate of formula H 
b) treating the product of Step a) above with potassium hexamethyldisilylazide followed by t-butyl acetate in THF to produce
the keto ester of formula I 
c) brominating the product from Step b) above to produce an intermediate of formula J 
d) cyclizing the product of Step c) above using potassium carbonate in DMF to produce enol of formula K 
The enol is then used to produce a compound of Formula 1 above.
Compounds of the invention are those prepared by the above process and especially those selected from:
2-(6-Methyl-benzo[1,3]dioxol-5-yl)4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester;
2-(2-Bromo-phenyl)-4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester;
2-(2-Chloro-phenyl)-4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester; and
2-(6-Chloro-benzo[1,3]dioxol-5-yl)-4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester.
The invention is also a process for the preparation of a compound of Formula 1 comprising:
a) reacting 2-methylanaline with benzenesulfonyl chloride to produce a compound of formula F 
b) treating the product of step a) above with n-butyl lithium and then with 3-methoxy-4,5-methylenedioxybenzaldehyde to produce a compound of formula M 
c) treating the product of step b) above with sodium hydride followed by methyl bromoacetate to produce a compound of formula N 
d) oxidizing the product of step c) above to produce the corresponding keto ester of formula O 
e) cyclizing the product d) above by treating with a base or a Lewis acid in an appropriate solvent to produce the corresponding ester of Formula 1 
Compounds of the invention are those prepared by the above process, especially 4-(7-Methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-o-tolyl-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester.
The invention is also a pharmaceutical composition of the compounds of Formula 1 above comprising a therapeutically effective amount of a compound of Formula 1 in admixture with a pharmaceutically acceptable carrier.
The compounds of the invention are useful in inhibiting elevated levels of endothelin comprising administering to a host in need thereof a therapeutically effective amount of a compound of Formula I in unit dosage form. They are also useful in treating subarachnoid hemorrhage, essential, renovascular, malignant and pulmonary hypertension, congestive heart failure, cerebral ischemia, or cerebral infarction.
In the compounds of Formula I, the term xe2x80x9calkylxe2x80x9d means a straight or branched hydrocarbon radical having from 1 to 12 carbon atoms unless otherwise specified and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, allyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, and dodecyl.
xe2x80x9cHalogenxe2x80x9d is fluorine, chlorine, bromine or iodine.
The term xe2x80x9calkoxyxe2x80x9d is an alkyl group as described above attached by oxygen to the rest of the molecule. Preferred alkoxy group are from 1 to 4 carbon atoms.
The host receiving a compound of the invention is a mammal, particularly a human.
The compounds of Formula I above may be prepared by several methods. These methods are illustrated in Schemes 1 through 3 and in a detailed manner by way of illustration in the example section of the specification.
Scheme 1 illustrates the general procedure for the preparation of the compounds of Formula 1. Synthesis of the intermediate A was described in U.S. Pat. No. 5,599,811. Reaction of methyl-2-(chlorosulfonyl)benzoate with an aniline in methylene chloride with 1 to 2 equivalents of pyridine and a catalytic amount of DMAP at room temperature overnight affords the sulfonamide A. The reaction can also be run using neat pyridine as the solvent. Alkylation of A was achieved by treatment of A with sodium hydride in DMF followed by reaction with methyl bromoacetate to give intermediate B. B can be converted to the key intermediate C via Claisen cyclization which were found to be promoted by at least two different catalysts. In condition I, the intermediate B in methylene chloride was added to a solution of 2 equivalents of TiCl4 in methylene chloride at xe2x88x9278xc2x0 C. followed by the treatment with 2.2 equivalents of triethyl amine. The reaction was quenched with 1N HCl followed by aqueous work up and recrystallization. The condition II is similar to condition I with the use of 1.2 equivalents of TiCl2(OTf)2 (Bull. Chem. Soc. Jpn, 1989;62:1917) followed by quench with pH 7 phosphate buffer and extractive workup. The cyclized enol C was treated with triflic anhydride in solvents such as methylene chloride in the presence of 2 to 5 equivalents of pyridine for 1 to 5 hours to afford the triflate D quantitatively. The coupling reaction was carried out between the triflate D and the corresponding boronic acid in DMF and toluene (ratio of 1:4) in the presence of 10 mole % tetrakis(triphenyl phosphine)palladium as the catalyst and 1 to 2 equivalents of potassium carbonate under reflux for 2 hours. The reaction mixture was filtered, and the product was purified by column chromatography or by recrystallization. The free acid of the Formula 1 was obtained by sponification of the ester with aqueous LiOH in THF/MeOH or in dioxane. The free acid can be converted, for example, to a sodium or potassium salts, which are much more soluble in H2O, by treating it with one equivalent of NaOH or KOH in MeOH followed by recrystallization.
Scheme 1a indicates an alternative assembly reaction for the formation of the 4-aryl benzothiazine derivatives exemplified by intermediate E. The coupling reaction was conducted by reacting an aryl zinc bromide, prepared by treating an aryl bromide with one equivalent of n-butyl lithium in THF followed by zinc bromide, with the intermediate D in the presence of 10% tetrakis(triphenylphosphine)palladium as the catalyst under reflux for 2 hours.
The Scheme 1 was further modified to replace a relatively expensive starting material, methyl-2-(chlorosulfonyl)benzoate, with phenyl sulfonyl chloride. The process (Scheme 1b) is proven to be more economical and workable in large scale. The sulfonamide F was formed by reacting the phenyl sulfonyl chloride with one equivalent of aniline under similar condition as described for A or using an equal amount of saturated sodium bicarbonate solution in THF. It was lithiated with n-BuLi at low temperature and quenched with CO2 to form the intermediate G in 92% yield. The acid G was then treated with acetic anhydride with a catalytic amount of methanesulfonic acid to form the intermediate H. This cyclic compound was treated with NaOMe followed by quenching with methyl bromoacetate to form B directly in 71% yield. 
Several compounds in the Examples were synthesized through a synthesis outlined in Scheme 2. Compound A, obtained as described in Scheme 1 was refluxed in xylene for 16 hours with 1 equivalent of pyridine and a catalytic amount of DMAP to afford the cyclic intermediate H. This compound was treated with potassium hexamethyldisilylazide followed by t-butyl acetate in THF at xe2x88x9278xc2x0 C. to yield the keto ester I. Bromination of I was carried out with NBS in carbon tetrachloride to obtain J which is in turn cyclized via treatment with potassium carbonate in DMF. The enol K was converted to the ester L with similar procedures illustrated in Scheme 1. Finally, the t-butyl ester was removed by treatment with TFA in methylene chloride at room temperature to give the free acid. 
Scheme 3 illustrates an alternative synthesis that has been successfully utilized in the synthesis of certain analogs. This is illustrated by the synthesis of Example 8. Intermediate F was treated with 2 equivalents of n-butyl lithium at xe2x88x9278xc2x0 C. to generate the dianion. Then a solution of 3-methoxy-4,5-methylenedioxybenzaldehyde in tetrahydrofuran was added to the reaction mixture. It was then warmed to 0xc2x0 C. over 1.5 hours. Aqueous work up afforded M which was treated with sodium hydride followed by methyl bromoacetate to give N. Jones oxidation of N was carried out in acetone to afford the keto ester O in 60% yield. The cyclization of O can be promoted by a base or a Lewis acid such as titanium bis-chloro-bis-triflate in an appropriate solvent such as methylene chloride. The ester E was purified by column chromatography to provide pure sample in 53% yield. The esters synthesized through this route were sponified according to procedures described in Scheme 1. 
Some of the compounds of Formula 1 are capable of further forming both pharmaceutically acceptable base salts. All of these forms are within the scope of the present invention.
Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,Nxe2x80x2-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge S M, et al., xe2x80x9cPharmaceutical Salts,xe2x80x9d Journal of Pharmaceutical Science, 1977;66:1-19).
The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
Certain of the compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention.
Certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the R or S configuration. The present invention includes all enantiomeric and epimeric forms as well as the appropriate diastereomeric mixtures thereof.
Test Protocols
The compounds of Formula 1 are valuable antagonists of endothelin. The tests employed indicate that compounds of the invention possess endothelin antagonist activity. Thus, the compounds were tested for their ability to inhibit [125I]-ET-1([125I]Endothelin-1) binding in a receptor assay. Selected compounds were also tested for antagonist activity by inhibition of ET-1 stimulated arachidonic acid release and ET-1 stimulated vasoconstriction. The following testing procedures were used (Doherty A M, et al., xe2x80x9cDesign of C-Terminal Peptide Antagonists of Endothelin: Structure-Activity Relationships of ET-1 [16-21, D-His16]xe2x80x9d, Bioorganic and Medicinal Chemistry Letters, 1993;3:497-502).
Radioligand Binding Assays
The following cultured cells were used in binding experiments: Ltkxe2x88x92cells expressing recombinant human ETAR (HETA), and CHO-K1 cells expressing recombinant human ETBR (HETB).
Membranes were prepared from cultured cells by lysing cells in cold lysis buffer (5 mM HEPES, 2 mM EDTA, pH 7.4) and homogenizing with a Dounce xe2x80x9cAxe2x80x9d homogenizer. The homogenate was centrifuged at 30,000xc3x97g for 20 minutes at 40xc2x0 C. Membrane pellets were suspended in cold buffer containing 20 mM Tris, 2 mM EDTA, 200 xcexcM Pefabloc, 10 xcexcM phosphoramidon, 10 xcexcM leupeptin, 1 xcexcM pepstatin at pH 7.4 and frozen at xe2x88x9280xc2x0 C. until use. Membranes were thawed and homogenized with a Brinkmann Polytron then diluted in tissue buffer containing 20 mM Tris, 2 mM EDTA, 200 xcexcM Pefabloc, and 100 xcexcM bacitracin (pH 7.4). Radioligand and competing ligands were prepared in binding buffer containing 20 mM Tris, 2 mM EDTA, and 0.1% BSA.
Competing binding assays were initiated by combining membranes, [125I]-ET-1 (40 pM) and the competing ligand in a final volume of 250 xcexcL and incubating for 2 hours at 37xc2x0 C. The assay was terminated by filtration over Whatman GF/B filters which were presoaked with 50 mM Tris, pH 7.4 containing 0.2% BSA and 100 xcexcM bacitracin. Nonspecific binding was defined as binding in the presence of 100 nM ET-1.
In Vitro Inhibition of ET-1 Stimulated Arachidonic Acid Release (AAR) in Cultured Rabbit Vascular Smooth Muscle Cells (ETA) by the Compounds of the Invention
Antagonist activity is measured by the ability of added compounds to reduce endothelin-stimulated arachidonic acid release in cultured vascular smooth muscle cells. [3H]Arachidonic Acid Loading Media (LM) is DME/F12+0.5% FCSxc3x970.25 mCi/mL [3H]arachidonic acid (Amersham). Confluent monolayers of cultured rabbit renal artery vascular smooth muscle cells were incubated in 0.5 mL of the LM over 18 hours, at 37xc2x0 C., in 5% CO2. The LM was aspirated and the cells were washed once with the assay buffer (Hank""s BSS+10 mM HEPES+fatty acid-free BSA (1 mg/mL)), and incubated for 5 minutes with 1 mL of the prewarmed assay buffer. This solution was aspirated, followed by an additional 1 mL of prewarmed assay buffer, and further incubated for another 5 minutes. A final 5-minute incubation was carried out in a similar manner. The same procedure was repeated with the inclusion of 10 xcexcL of the test compound (1 nM to 1 xcexcM) and 10 xcexcL ET-1 (0.3 nM) and the incubation was extended for 30 minutes. This solution was then collected, 10 mL of scintillation cocktail was added, and the amount of [3H]arachidonic acid was determined in a liquid scintillation counter.
In Vitro Antagonism of ET-1 Stimulated Vasoconstriction (VERAA) in the Rabbit Femoral Artery (ETA) and Sarafotoxin 6C Stimulated Vasoconstriction in the Rabbit Pulmonary Artery (ETB)
Male New Zealand rabbits were killed by cervical dislocation and exsanguination. Femoral and pulmonary arteries were isolated, cleaned of connective tissue, and cut into 4-mm rings. The endothelium was denuded by placing the rings over hypodermic tubing (32 gauge for femoral rings and 28 gauge for pulmonary rings, Small Parts, Inc., Miami, Fla.) and gently rolling them. Denuded rings were mounted in 20 mL organ baths containing Krebs-bicarbonate buffer (composition in mM: NaCl, 118.2; NaHCO3, 24.8; KCl, 4.6; MgSO4 7H2O, 1.2; KH2PO4, 1.2; CaCl2 2H2O; Ca-Na2 EDTA, 0.026; dextrose, 10.0), that was maintained at 37xc2x0 C. and gassed continuously with 5% CO2 in oxygen (pH 7.4). Resting tension was adjusted to 3.0 g for femoral and 4.0 g pulmonary arteries; the rings were left for 90 minutes to equilibrate. Vascular rings were tested for lack of functional endothelium (ie, lack of an endothelium-dependent relaxation response to carbachol (1.0 nM) in norepinephrine (0.03 nM) contracted rings. Agonist peptides, ET-1 (femoral), and S6c (pulmonary), were cumulatively added at 10-minute intervals. The ET antagonists were added 30 minutes prior to adding the agonist.
Inhibition of bET-1 Induced Pressor Effect in Rat
Male Sprague-Dawley (Charles River Laboratories, Kingston, Ontario, Canada) weighing 250 to 350 g are anesthetized (Inactin, 120 mg/kg, I.P.) and acutely instrumented with a carotid artery catheter to monitor arterial blood pressure and with a jugular vein catheters to administer intravenous drugs. Once instrumented, the rats are ganglionic-blocked with mecamylamine (1.25 mg/kg, I.V.) to prevent hemodynamic reflexes and then challenged with big endothelin-1 (bET-1) (1.0 nmol/kg, I.V.). The peak arterial pressor response in rats pre-treated with selected compounds of invention compared to vehicle-treated rats is used to determine activity expressed as % inhibition. For I.V. activity selected compounds of invention was dose 10 minutes before the bET-1 challenge, and for oral activity selected compounds of invention was administered via oral gavage 8 or 24 hours before the bET-1 challenge.
For example, Example 10 is a potent inhibitor (IC50=0.6 nM) of ET-1 binding to human ETA receptors. It also has potent functional antagonist activity in human pulmonary artery smooth muscle cells blocking ET-1 induced Ca++ transients via the ETA receptor (IC50=0.2 nM). It exhibits antagonism to ET-1 stimulated vasoconstriction in rabbit femoral artery with pA2 value of 7.7. In vivo, ETA blockade with oral Example 10 was demonstrated through inhibition of bET-1 pressor response in the rat (10 mg/kg produced 44% inhibition at 8 hours postdose and 30 mg/kg produced a 48% inhibition at 24 hours postdose) and dog (15 mg/kg produced 25% inhibition at 24 hours postdose).
Oral Example 10 inhibited acute hypoxic pulmonary hypertension in the rat in a dose dependent manner with the ED50=0.8 mg/kg (the plasma EC50=0.046 xcexcg/mL). Example 10 was well absorbed orally both in rat and dog. Terminal elimination txc2xd""s were determined to be 8.5 and 2.2 hours in rat and dog, respectively, and bioavailability is 77% and 100% in rat and dog.
The compounds of the present invention can be prepared and administered in a wide variety of oral and parenteral dosage forms. Thus, the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compounds of the present invention can be administered by inhalation, for example, intranasally. Additionally, the compounds of the present invention can be administered transdermally. It will be obvious to those skilled in the art that the following dosage forms may comprise as the active component, either a compound of Formula 1 or a corresponding pharmaceutically acceptable salt of a compound of Formula 1.
For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.
In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term xe2x80x9cpreparationxe2x80x9d is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions. For parenteral injection liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired.
Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, table, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
The quantity of active component in a unit dose preparation may be varied or adjusted from 0.1 mg to 100 mg preferably 0.5 mg to 100 mg according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.
In therapeutic use as antagonists of endothelin, the compounds utilized in the pharmaceutical method of this invention are administered at the initial dosage of about 0.01 mg to about 100 mg/kg daily. A daily dose range of about 0.01 mg to about 10 mg/kg is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
The following nonlimiting examples illustrate the preferred methods for preparing the compounds of the invention.
Following are prepared according to Scheme 1.
Methyl 2-(chlorosulfonyl)benzoate (23.5 g, 0.1 mol) was dissolved in 150 mL of anhydrous pyridine in a 500 mL flask. Catalytic amount of DMAP (4-dimethylaminopyridine) (0.04 eq) was added. 2-Trifluoromethylaniline (20 g, 0.12 mol) was added to the reaction mixture, and it was allowed to stir at room temperature overnight. Pyridine was removed, and the residue was diluted with 250 mL of EtOAc. It was then washed with IN HCl solution followed by brine. After drying with MgSO4, the EtOAc was removed in vacuo to give a thick red oil. The crude product was recrystallized from hexane/Et2O to give the product 1A as white crystals (17.63 g, 49% yield).
Analysis calculated for C15H12F3N1O4S1: C, 50.14; H, 3.37; N, 3.90. Found: C, 50.02; H, 3.28; N, 3.82.
MS (CI) M/E 359.8 (M+)
To the suspension of NaH (60% in mineral oil, 2.0 g, 50 mmol) in 45 mL of DMF at 0xc2x0 C. was added a solution of 2-(2-trifluoromethyl-phenylsulfamoyl)-benzoic acid methyl ester (12.0 g, 33.3 mmol) in 22 mL of DMF dropwise. After about 10 minutes, 11.22 g (66.8 mmol) of methyl bromoacetate was added, and reaction solution was warmed to room temperature in 3 hours. The light yellow reaction mixture was poured into ice water. The mixture was then extracted with EtOAc 3xc3x97(xcx9c400 mL) and washed with brine. The organic layer was dried over MgSO4 and evaporated down to a sick light orange oil. White crystals were formed upon treatment with Et2O and hexane. It was collected and dried over the vacuum oven to give pure 2-[methoxycarbonylmethyl-(2-trifluoromethyl-phenyl)-sulfamoyl]-benzoic acid methyl ester (13.85 g, 96%).
MS (Cf) M/E 431.7 (M+)
Alternative procedure: To dimethylformamide (220 mL) was added 2-(2-trifluoromethyl-phenylsulfamoyl)-benzoic acid methyl ester, (8.0 g, 22.2 mmol). After cooling to 0xc2x0 C., a solution of potassium hexamethyl-disilazide (0.5 M, 44.5 mL) in toluene was added. Methyl bromoacetate (4.32 mL, 45.6 mmol) was added, followed by stirring at 70xc2x0 C. overnight. The solvent was evaporated in vacuo, and the residue was suspended in ethyl ether and washed with saturated sodium bicarbonate, brine, and 1N citric acid solutions. The ether was dried over anhydrous magnesium sulfate, filtered, and evaporated to give a crystalline solid, 5.59 g, 58% yield. NMR spectra and elemental analysis were consistent with the structure.
Compound 1B can also be synthesized using procedures outlined in Scheme 1b with benzenesulfonyl chloride. Following is the experimental detail:
a. Preparation of 1F: N-(2-Trifluoromethyl-phenyl)-benzenesulfonamide
Benzenesulfonyl chloride (400 g, 2.26 moles) was dissolved in dichloromethane (2 L). To this was added o-trifluoromethylaniline (250 mL, 320 g, 1.99 mol), pyridine (222 mL), and DMAP (7.5 g). The resulting solution was stirred at room temperature for 3 days (72 h), then heated to reflux for 1 hour. The cooled solution was then concentrated on the rotary evaporator, with the bath temperature gradually raised to 70xc2x0 C. The residue was taken up in ether (3 L) and 1N HCl (1.5 L). The ether layer was separated, washed 2xc3x97500 mL with 1N HCl, 1xc3x971 L with brine, 1xc3x971 L with saturated sodium bicarbonate, and once more with 1 L of brine. It was then dried over magnesium sulfate, filtered with suction, and concentrated on the rotary evaporator without heat. The product formed fine crystals as the ether evaporated, and when most of the ether was gone, the crystals were slurried with hexane (2 L), collected with suction and dried. Yield 571 g, 84%.
b. Preparation of 1G: 2-(2-Trifluoromethyl-phenylsulfamoyl)-benzoic acid
A 22-L flask was equipped with an overhead stirrer, nitrogen inlet and thermocouple, and placed in an acetone bath. The flask was charged with 1F (544.5 g, 1.807 mol) and THF (10 L, from freshly opened bottles) and stirred. When a solution had formed, dry ice was added to the bath until the temp was  less than xe2x88x9270xc2x0 C. n-Butyllithium (2.4 L of 1.6M, 3.84 mol) was added in portions, keeping the temperature below xe2x88x9255xc2x0 C. (30 minutes). The reaction mixture was then stirred at xe2x88x9270xc2x0 C. to xe2x88x9272xc2x0 C. for 3 hours, then bone-dry carbon dioxide was bubbled in, keeping the temperature below xe2x88x9255xc2x0 C. This was continued until no further exotherm was observed, then about 10 minutes longer. The mixture was then warmed to room temperature over 1 hour, transferred to a rotary evaporator, and stripped at 50xc2x0 C. until no more THF came off. The residue was dissolved in water (5 L), and the basic solution was washed with 3xc3x971 L ether. The solution was then acidified by addition of 330 mL concentrated HCl. The acid product was extracted from the aqueous layer with 3xc3x971 L ether. The combined ether layers were dried over magnesium sulfate, filtered, and stripped on the rotary evaporator. The solid residue was broken up and agitated with hot tap water (4 L, at about 60xc2x0 C.) for 20 minutes, then collected with suction and dried in vacuo (1 mm Hg) for 24 hours. Yield: 575 g, 92%. This material contained traces amount of impurities, which were removed in the next step.
c. Preparation of 1H: 1,1-Dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[d]isothiazol-3-one
Compound 1G (300 g, 0.876 moles) was slurried in acetic anhydride (350 mL). Methanesulfonic acid (3 drops) was added, and the resulting mixture was heated to reflux for 20 minutes. The resulting solution was allowed to stand without further heating for 30 minutes, then the apparatus was rearranged for distillation, and 190 mL of distillate was removed. The pot residue had begun to precipitate solids even at the boiling point, and 20 mL of the distillate was added back to improve workability of the mixture. The solids were broken up carefully as the mixture cooled, and heptane (xcx9c100 mL) was added, followed by ether (xcx9c50 mL). The product was collected with suction, and washed with several small portions of ether (xcx9c150 mL total). Yield 266 g, 94%.
d. Preparation of 1B from 1H
Compound 1H (244 g, 0.745 mol) was added to a solution of sodium methoxide freshly prepared from 19 g (0.83 mol) of sodium and 600 mL methanol in a 3N 3-L round bottom flask equipped with a reflux condenser under nitrogen. The mixture was stirred for 30 minutes without further heating, at which point a greenish-yellow solution had formed. Methyl bromoacetate (83 mL, 137 g, 0.9 moles) was added, and the mixture was stirred without heat for 15 minutes, then heated to reflux for 90 minutes. The mixture was then cooled to 0xc2x0 C., and the resulting ppt was collected with suction, washed 2xc3x9775 mL with ether, then 4xc3x97200 mL with water. The product was dried in vacuo (1 mm Hg) 2 hours at 60xc2x0 C. Yield 228 g, 71%.
In a 250 mL dry RB flask, 4.50 mL of TiCl4 (40.8 mmol) was dissolved in 100 mL of CH2Cl2 and was cooled to xe2x88x9230xc2x0 C. A solution of 2-[methoxycarbonyl-methyl-(2-trifluoromethyl-phenyl)-sulfamoyl]-benzoic acid methyl ester (8.0 g, 18.54 mmol) in 50 mL CH2Cl2 was added dropwise. The resulting solution was kept at xe2x88x9230xc2x0 C. for 30 minutes and was then cooled to xe2x88x9278xc2x0 C. Et3N (7.75 mL, 55.6 mmol) in 50 mL CH2Cl2 was slowly syringed in resulting a dark red solution. The reaction mixture was stirred at xe2x88x9278xc2x0 C. for 4 hours and was then poured into a 1-L flask containing 250 mL of Et2O, 100 mL concentrated HCl, and 100 mL of H2O. The mixture was stirred at room temperature overnight. The layers were separated, and the organic layer was washed with brine and dried over MgSO4. Upon evaporation of the solvent, light yellowish crystals of 4-hydroxy-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester were formed. The product was collected through filtration and dried in oven to provide 2.2 g, 86% yield.
Analysis calculated for C15H12F3N1O4S1: C, 50.12; H, 3.74; N, 3.25; Found: C, 50.04; H, 3.76; N, 3.11.
To dichloromethane (150 mL) was added 4-hydroxy-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester, (13.1 g, 32 mmol). After cooling to 0xc2x0 C., pyridine (12.8 mL, 158 mmol) was added, followed by trifluoroacetic anhydride (10.7 g, 38 mmol). The mixture was stirred 30 minutes and allowed to warm to 25xc2x0 C., then was poured into ethyl acetate (200 mL). The mixture was washed with brine, dried over anhydrous magnesium sulfate, filtered, and evaporated to a solid. Recrystallization from ethyl acetate and ethyl ether gave a crystalline solid which was filtered and dried in vacuo giving a solid, 15.5 g, 92% yield.
1H NMR (400 MHz, CDCl3): xcex47.25-7.9 (m, 8 H), 3.78 (s, 3 H).
1. Preparation of 3,5-dimethoxy-phenyl-boronic acid
3,5-Dimethoxychlorobenzene (14.0 g, 81 mmol) in 10 mL of THF was added dropwise into a refluxing solution of 2.5 g (100 mmol) of Mg turning in 15 mL of THF with 2 drops of dibromoethane. After the addition, the reaction mixture was refluxed for 48 hours. The Grignard solution was then added into a solution of 40.0 g (380 mmol) of trimethyl borate in 300 mL of THF at 0xc2x0 C., stirring at room temperature overnight. The reaction solution was diluted with 1N HCl (400 mL) and ethyl acetate (300 mL). The organic layer was separated and was washed with 1N HCl (50 mL), water, brine, dried with MgSO4, concentrated down, and recrystallized in ethyl acetate and ether to give 3.15 g (17.5 mmol, 22% yield) boronic acid.
1HNMR (300 MHz, CDCl3): xcex46.48 (s, 1H), 5.14 (b, 2H), 6.96 (s, 2H).
2. Preparation of 4-(3,5-dimethoxy-phenyl)-2-(2-trifluoromethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester (1E)
To dimethylformamide (18 mL) was added 1,1-dioxo-4-(trifluoro-methanesulfonyloxy)-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester, (1.60 g, 3.0 mmol), palladium tetrakis(triphenyl phosphine) (0.2 g, 0.17 mmol), potassium carbonate (0.7 g, 6.0 mmol), 2-methoxy-phenyl-boronic acid (0.88 g, 6.0 mmol), and toluene (50 mL). The mixture was heated to 100xc2x0 C. for 30 minutes and was filtered through a pad of Celite. The filtrate and EtOAc washing solution was washed three times with saturated sodium bicarbonate solution, brine, and dried over charcoal and anhydrous magnesium sulfate. The suspension was filtered and stripped to an oil which was resuspended in ethyl acetate and ethyl ether. Insoluble material from this mixture was filtered off, and the filtrate was concentrated in vacuo to an oil. The residue was chromatographed on silica gel eluted with a mixture of ethyl acetate and hexane (25:75). The appropriate fractions were concentrated in vacuo and crystallized from ethyl ether giving a solid 1.29 g, 83% yield.
Analysis calculated for C25H20F3N1O6S1: C, 57.80; H, 3.88; N, 2.70; Found: C, 57.56; H, 3.86; N, 2.66.
MS: m/e 519 (M+)